Biologic composition and method of manufacture

ABSTRACT

A biological composition has a mixture of mechanically selected allogeneic biologic material derived from bone marrow. The mixture has non-whole cellular components including vesicular components and active and inactive components of biological activity, cell fragments, cellular excretions, cellular derivatives, and extracellular components. The mixture is compatible with biologic function.

RELATED APPLICATIONS

This invention is a division of co-pending U.S. application Ser. No.14/683,221 filed on Apr. 10, 2015 entitled, “Biologic Composition AndMethod Of Manufacture”.

TECHNICAL FIELD

This invention is a tissue regenerative biological composition. Morespecifically, a composition at least in part formed from bone marrow anda method of manufacture and use of said composition.

BACKGROUND OF THE INVENTION

In the area of tissue regeneration or repair, the use of stem celltherapy has been widely touted.

Often, these inventions describe isolating the stem cells, purifying andculturally expanding mesenchymal stem cells. In U.S. Pat. No. 5,837,539,entitled “Monoclonal Antibodies For Human Mesenchymal Stem Cells”,Arnold Caplan et al. reported that the cells are preferably culturallyexpanded, but suggest it is possible to use the stem cells withoutculture expansion. Caplan also describes a way to isolate stem cells.

A major technological hurdle to producing a safe allogeneic compositionwith viable cells has been the need to approach a fraction of riskapproaching zero by removing all antigenic properties that lead toinflammation factors in a separation to yield only a certain stromalcell type. This has proven both difficult and degrading the quantity ofviable cells that can be effectively harvested.

The present invention has yielded a biological composition that is safeand achieves high yields of viable stromal cells and does so in a methodthat allows the resultant mixture to be recovered in a non-expanded andnon-differentiated way from bone marrow wherein the mixture unexpectedlyexhibits increased CD105 and STR01 markers at time of use when comparedto the quantity at the time of actual processing. This evidences amaintenance of viable cells in the dose, an increase in mesenchymalcells in the dose and a legacy or memory of the lineages from where thecells came which retain the ability to differentiate into new tissueforms other than bone.

These and other benefits of the present invention and the method ofpreparing it are described hereinafter.

SUMMARY OF THE INVENTION

A biological composition has a mixture of mechanically selectedallogeneic biologic material derived from bone marrow. The mixture hasnon-whole cellular components including vesicular components and activeand inactive components of biological activity, cell fragments, cellularexcretions, cellular derivatives, and extracellular components. Themixture is compatible with biologic function.

The mixture of mechanically selected material derived from bone marrowfurther can have non-expanded whole cells. The biological compositionpreferably has bone particles. The bone particles can be added to themixture derived from bone marrow. The bone particles include a mixtureof cortical bone particles and cancellous bone particles.

The combination of non-whole cell components with a select number of thenon-expanded cells sustains pluripotency in the cells. The select numberof the non-expanded cells includes differentiated committed cells andnon-differentiated and non-committed cells.

In a preferred embodiment, the biological composition is predisposed todemonstrate or support elaboration of active volume or spatial geometryconsistent in morphology with that of endogenous bone. The biologicalcomposition extends regenerative resonance that compliments or mimicstissue complexity. The mixture is treated in a protectant orcryoprotectant prior to preservation or cryopreservation. The protectantor cryoprotectant creates a physical or electrical or chemical gradientor combination thereof for tissue regeneration. The gradient can have aphysical characteristic of modulus or topography. The gradient can havea chemical characteristic of spatially changing compositions of densityor species of functional molecules. Also, the gradient can have anelectrical characteristic of charge based or pH based or electronaffinities that confer metastability in biologic potential.

The bone marrow mixture which is derived from a cadaver hasseparation-enhanced cell vitality including one or more of thefollowing: separating the cells heightens their vitality, reversing“arrest” of donors, responsive molecular coupling, matrix quest inneutralizing inflammation or satience by balancing stimulus for repair.The protectant or cryoprotectant is a polyampholyte. The regenerativeresonance occurs in the presence or absence of a refractory response.When using a cryoprotectant, the cryopreservation occurs at atemperature that is sub-freezing wherein the cryopreservationtemperature is from 0 degrees C. to −200 degrees C.

The biological composition's non-whole cellular component also caninclude organelle fragments and the active and inactive components ofbiological activity which can also include extants of the humanmetabolome.

A method of making a biological composition of the present invention hasthe steps of: collecting, recovering and processing bone marrow from acadaver donor; mechanically separating the cellular or non-cellularcomponents or a combination thereof of bone marrow from cadaverous bone;concentrating by centrifugation and filtering; separation by densitygradient centrifugation; collecting cellular or non-cellular componentsor a combination thereof of predetermined density; washing the cellularor non-cellular components or a combination thereof to create themixture; quantifying cell concentration not to exclude zero; suspendingto a predetermined concentration in a polyampholyte cryoprotectant;freezing the mixture at a predetermined controlled rate; and packaging abone blend having particles in the size range of 100 to 300 μm ofdemineralized cortical bone, mineralized cortical bone and mineralizedcancellous bone either within the mixture or separate. These particlesize ranges can vary higher or lower depending on the application. Atthe time of use, the mixture is thawed by immersion in a warm water bathfor 2-3 minutes at 37 degrees C. It is diluted in saline withoutspinning; and then the diluted mixture, with or without the bone blendbeing intermixed, can be implanted by packing, injection, scaffolding orany other suitable means into a patient.

DEFINITIONS

DNase—deoxyribonuclease is any enzyme that catalyzes the hydrolyticcleavage of phosphodiester linkages in the DNA backbone, thus degradingDNA.

DMEM, DMEM/LG—Dulbecco's Modified Eagle Medium, low glucose. Sterile,with: Low Glucose (1 g/L), Sodium Pyruvate; without: L-glutamine, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

DPBS—Dulbecco's Phosphate Buffered Saline.

CBT-MIXER—Mixing blade for Cancellous Bone Tumbler Jar.

Cold Media—Media used during the preparation of vertebral bodies forinitial processing.

Cryopreserved—Tissue frozen with the addition of, or in a solutioncontaining, a cryoprotectant agent such as glycerol ordimethylsulfoxide.

Freeze Dried/Lyophilized—Tissue dehydrated for storage by conversion ofthe water content of frozen tissue to a gaseous state under vacuum thatextracts moisture.

Normal Saline—0.9% Sodium Chloride Solution.

Packing Media—Media used during initial processing and storage of theprocessed vertebral bodies prior to bone decellularization.

PBS—Phosphate Buffered Saline.

Processing Media—Media used during bone decellularization that maycontain DMEM/Low Glucose no phenol red, Human Serum Albumin, Heparin,Gentamicin and DNAse.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described by way of example and with reference tothe accompanying drawings in which:

FIG. 1 shows a photograph of a cut vertebral body taken from a spine ofa cadaver donor.

FIG. 2 shows a photograph of the vertebral body after being cut intocubic pieces and immersed in a packing media.

FIG. 3 shows a photograph of the bulk bone material after being groundand immersed in packing media and placed in a jar for later tumbling.

FIG. 4 shows a photograph of the jar with a CBT-Mixer connected to atumbler.

FIG. 5 is a photograph of an exemplary sieve device having sieves sizedto separate the solid material.

FIG. 6 shows a photograph of two 50 ml vials, the one on the left beingprior to centrifuging with the Ficoll that is commercially available atthe bottom and the material above it. The 50 ml vial on the right isafter centrifuging showing the cell interface layer.

FIG. 7 is a photograph showing the four tumbling steps 1-4 by exemplarycollection and Ficoll separation of the decanted fluids, the fluid intumble 1 being completely discarded to remove unwanted debris.

FIG. 8A is a chart showing the percent recovery at 6 months aftercryofreezing the mixture of 1 ml at 1.1×10⁶ cells and thawing.

FIG. 8B is a chart showing the viability at 6 months after cryofreezingand thawing.

FIG. 8C shows a chart at 6 months of MSC markers by percentage of cells.

FIGS. 9A and 9B are photographs of cells thawed from a single sample andplaced in media at 37 degrees C. overnight evidencing cell viability.

FIG. 10 is a representative photograph of the final packaging.

FIG. 11 is a photograph showing the ground bone.

DETAILED DESCRIPTION OF THE INVENTION

With reference to the present invention which is a tissue regenerativebiological composition 100 made from bone marrow 200, it is believedbest understood by the methods used to process and recover thebiological composition, as illustrated in the FIGS. 1-6.

The first steps are to collect, recover and process bone marrow 200 froma cadaver donor. To do this, the spine is removed aseptically from thecadaver and the resultant spine segment is covered by cold media. Thecold media has 0.5 ml of Heparin; 10,000 units/ml per 500 ml of DMEM.DMEM is a sterile solution with low glucose (1 g/L), Sodium Pyruvate;without L-glutamine, or HEPES. This cold media is used for packaging thespine segments for later processing. At this point the spine segmentincludes a plurality of vertebral bodies 202. The clinical technicianmust remove as much soft tissue as possible and cut each vertebral body202 with a saw. These vertebral bodies 202, once cleaned, of alladherent soft tissue around the cortical surfaces will look as shown inFIG. 1.

Once a cleaned vertebral body 202 is obtained, the next step involvescutting each vertebral body 202 into pieces, each piece 204 roughly 1cm³. The cut pieces 204 being immersed in a packing media 400. Theexemplary packing media can be DMEM with 0.5 mlHeparin and 1.25 ml ofDNAse added.

Once all the vertebral bodies 202 have been cut, the pieces 204 aretaken to the bone grinder. The bone is ground into 4-10 mm pieces usingpacking media 400 to help the pieces go through the grinder. The groundbone 206 (bulk cortical-cancellous crushed) and all of the packing media400, estimated volume of 500 ml are transferred into a jar 300 where0.5-1.0 ml of Gentamicin is added to the jar 300 with ground bone 206and packing media 400. At this point, the crushed bone 206, includingcellular soft marrow 200, is intermixed.

The step of mechanically separating these cellular components of bonemarrow 200 from the cadaverous bone is next performed. Transferring thebulk cortical-cancellous bone chips into a new jar with a CBT-Mixer inthe jar. The bulk cortical-cancellous bone chips 206 will go throughfour cycles as summarized in the table below. Each cycle, after cycle 1,contains three steps using a bone tumbler 500 and sieve set 600. Thesieve set 600 has screens 602 of various sizes, for example 500 μm and180 μm, as shown in FIG. 5.

Step Cycle 1 Cycle 2 Cycle 3 Cycle 4 Bone 30 minutes. 30 minutes 30minutes 30 minutes Tumbler Using Using Using Using 500 mL 500 mL 500 mL400 mL Processing Processing Processing Processing Media Media MediaMedia Sieve Use the 500- Use the 500- Use the 500- Use the 500- Set μmand the μm, 180-μm μm, 180-μm μm, 180-μm bottom and bottom and bottomand bottom pan sieve. pan sieve. pan sieve. pan sieve. Discard CollectCollect Collect decanted decanted decanted decanted fluid. fluid. fluid.fluid. Centri- N/A Use decanted Use decanted Use decanted fuge fluid.fluid. fluid.

In cycle 1, the decanted fluid 210 is discarded. To best understandthis, an exemplary FIG. 7 shows conical tubes with the decanted fluidsafter each cycle followed by Ficoll separation. Tumble 1 or Cycle 1 hasmost of the unwanted cells and debris as evidenced by its dark and redappearance whereas each subsequent cycle 2, 3 and 4 are progressivelycleared. This FIG. 7 is only to illustrate the effects of multipletumbles 1-4 and the value in discarding the decanted liquid 210 afterthe first tumble 1.

After each subsequent sieving of the bulk bone material 206, thedecanted fluid 212, 214, 216 containing the mixture with whole cells iscollected and put into a collection jar. When the next three cycles arecomplete and the decanted fluid is all placed in the collection jarcomingling the fluids 212, 214 and 216 to form a decanted fluid 220.Then the centrifugation of the combined decanted fluid 220 occurs byplacing the fluid 220 in a number of 250 ml conical tubes using a 100 mlpipette. The centrifuge is programmed to 280×g for 10 minutes at roomtemperature, preferably about 20 degrees C. The fluid 220 is passedthrough a blood filter to further remove any bone or spicules or clumpsfrom the suspended cells. This completes the step of centrifuging andfiltering. At this point, the mixture including whole cells 240 has beenseparated from the soft marrow tissue 200 and the remaining cancellousand cortical bone is discarded.

After this as shown in FIG. 6, the step of separating the cells 240 by adensity centrifugation occurs. The mixture including whole cells 240 isplaced in 50 ml conical tubes 20 with Ficoll 800 and undergoes aFicoll-Paque separation under centrifugation wherein a cell densitygradient is established by spinning at 400×g for 30 minutes at roomtemperature, preferably about 20 degrees C. The mixture includescellular or non-cellular components or a combination thereof. All fluid211 above the interface is removed and the interface 230 including thedesired components which can include whole cells 250 is then collectedusing a 5 ml pipette and transferred into new 50 ml conical tubesensuring no tube has more than 10 ml. Then the volume is brought to 50ml by adding DPBS and centrifuged at 400×g for 5 minutes at roomtemperature, preferably about 20 degrees C. and the supernatant isremoved leaving a pellet. Each 50 ml tube is then filled up to 50 mlwith DPBS to resuspend the pellet. Another centrifugation occurs and thesupernatant is removed and the remaining pellet is resuspended using theprocess media with no antibiotics. The suspension is then used toresuspend all the pellets in remaining tubes. The suspension volume isbrought to 50 ml by adding processing media with no antibiotic. Then thesuspension can be strained using a 100 μm cell strainer if any visualclumping is seen. These steps effectively wash the cells 250, ifpresent, and the non-cellular components. A representative sample isthen counted. The remaining, or a portion thereof, of the cellular ornon-cellular components or a combination thereof is centrifuged andresuspended in the desired protectant after which it's placed in vialsholding 1 ml.

In the preferred embodiment, this results in 1.1×10⁶ cells per ml, butcould cover any concentration from zero to less than 5.0×10⁶ cells perml depending on the desired concentration wanted per cc.

Once the cell count is established and each 1 ml suspension isestablished or quantified, the material is taken and suspended in apredetermined concentration of a polyampholyte cryoprotectant or anyother suitable alternative protectant. When using the cryoprotectant, afreezing of the mixture at a predetermined control rate is required.Ideally, the application of a cryoprotectant coats each cell 250 andprovides a protective coating to keep the cell viable during thefreezing process. While the techniques for cryopreservation are wellknown, the present invention after being frozen has demonstratedremarkably unexpected results.

When thawed and a cell count is preformed after manufacture, the cellviability is 80 percent. Thawing is in a water bath warmed to 37 degreesC. for 2-3 minutes. After storage for 6 months, the cell viability is91.0 +/−3.8%. The percent recovery from freeze at 6 months thaw is 82.8+/−7.2%. The inventors have noted that the recovery count is lower thanthe viability to the lysis of undesirable GlycoA+cells during freeze, awell-known occurrence. The unlysed desirable cells were viable at 91.0%.The inventors would also like to note that while thawed cells aregenerally suspended in FDS-supplemented media and spun, to bettersimulate how the product is actually used the cell recovery at sixmonths was thawed and suspended in 3 ml of saline yielding a 4 mlsuspension and that was not spun, but measured directly to simulate areal use injection. This allowed the cryopreservative to moreeffectively demonstrate that actual count of viable cells a patientwould expect to receive and provides one explanation for this remarkableviability result. As shown in FIGS. 9A and 9B, the cells 250 are shownunder magnification. In the cells at 6 months thaw the percent ofpositive cells for MSC markers, specifically CD105 and STR01+ are 52percent and 74 percent respectively. These indicate the majority ofcells are non-differentiated and directionally favorable for new boneformation.

Once the mixture is completed, the method can include additional steps.This leads to the use of a bone blend 102 shown in FIG. 10, preferablyfrom the same vertebral bone or at least bone from the same donor.

When the mixture is prepared, it can have whole cells or even no wholecells, but will have the mechanically selected non-whole cellularcomponents including vesicular components and active and inactivecomponents of biological activity, cell fragments, cellular excretions,cellular derivatives, and extracellular components.

In one embodiment, the composition includes the whole cells in themixture. In that embodiment, it is possible to provide bone particleswith the mixture either in the mixture or separately to be combined atthe time of use.

In one embodiment, the bone is ground to a particle size of 100-300 μm,see FIG. 11. The bone mixture has 1.5 cc of mineralized cancellous bone104, 1.5 cc of mineralized cortical bone 105 and 2.0 cc of demineralizedcortical bone 106 yielding 30 percent, 30 percent and 40 percentrespectively of the total 5 cc (5 gram) of bone material 102. The rangescoincide with the 1 cc of mixture when resuspended in 3 cc of saline toprovide a bone particle and mixture for implantation, which can be bypacking, injection, scaffolding or any other suitable means, into apatient in a fracture healing procedure, by way of example.

Other ranges of bone particle sized and mixture can be employeddepending on the application which, in this example, was boneregeneration. Lower volumes and cell counts may be more suited for lessintrusive bone repairs or more if larger if larger amounts of materialare needed as in a hip defect or repair.

Variations in the present invention are possible in light of thedescription of it provided herein. While certain representativeembodiments and details have been shown for the purpose of illustratingthe subject invention, it will be apparent to those skilled in this artthat various changes and modifications can be made therein withoutdeparting from the scope of the subject invention. It is, therefore, tobe understood that changes can be made in the particular embodimentsdescribed, which will be within the full intended scope of the inventionas defined by the following appended claims.

What is claimed is:
 1. A method of preparing a biological compositionfor use comprises the steps of: collecting, recovering and processingbone marrow from a cadaver donor; mechanically separating cellular andnon-cellular components of bone marrow from cadaverous bone;concentrating by centrifugation and filtering; separation by densitygradient centrifugation; collecting cells or non-cellular components orcombinations thereof of predetermined density; washing the cells ornon-cellular components or combinations thereof to create a mixture;quantifying cell concentration not to exclude zero; suspending to apredetermined concentration in a polyampholyte cryoprotectant; freezingthe mixture at a predetermined controlled rate; and packaging a boneblend having particles in the size range of 100 to 300 μm ofdemineralized cortical bone, mineralized cortical bone and mineralizedcancellous bone either within the mixture or separate.
 2. The method ofpreparing the biological composition for use of claim 1 by the steps of:thawing the mixture; and implanting the diluted mixture with or withoutthe bone blend being intermixed by packing, injection or any othersuitable means into a patient.
 3. The method of preparing the biologicalcomposition for use of claim 2 wherein the step of thawing the mixtureoccurs at a temperature of 37 degrees C. for 2 to 3 minutes in a warmwater bath.
 4. The method of preparing the biological composition foruse of claim 2 further comprises the step of: diluting the thawedmixture in saline without spinning.
 5. The method of preparing thebiological composition for use of claim 3 further comprises the step of:diluting the thawed mixture in saline without spinning.